SUMMARY: Alzheimer's disease (AD) is the most devastating age-related disease due to its damage to cognitive and motor capacity, heavily care-dependent nature, and lack of early diagnostics and effective therapeutics. The first step toward its cure is to discover early-stage molecular markers based on its fundamental pathogenesis: accumulation of protein aggregates in plaques and chronic proinflammation. The main biochemical regulatory mechanism governing these two cellular events is post-translational modification of proteins by ubiquitin (Ub) and ubiquitin-like proteins (Ubls). Circulating immune cells in blood and cerebrospinal fluid (CSF) provide a means of liquid biopsy to monitor protein degeneration in the brain. We hypothesize that the signature of the Ub and Ubl immune proteome serves as a marker for AD. However, the precise information encoded in these Ub/Ubl modification sites is poorly understood. This is mainly due to a lack of reagents to selectively isolate Ub- or Ubl-conjugated peptides from protease-treated cell lysates. We will develop methods to allow the specifically isolation and identification of Ub or Ubl conjugation sites at the proteome level. This will be accomplished by developing a novel affinity matrix to specifically enrich the ubiquitin remnants generated from protease digestion, resulting in optimal quantification by mass spectrometry analysis. Using this technology, we will identify the bona fide pan-ubiquitylation patterns of the immune proteome as a pathological fingerprint for neurodegeneration. The success of this project will provide the first comprehensive pan-ubiquitin immune proteome to monitor and diagnose AD.